Investigating the regulation of fos during embryonic wound healing and morphogenesis in Xenopus

Extracellular signal-related kinase (ERK) regulates gene expression through substrate phosphorylation. The conserved transcriptional repressor Capicua (CIC) is negatively regulated via ERK-mediated phosphorylation. Both fibroblast growth factor (FGF) signalling and embryonic wounding activate ERK, however the contribution of CIC to transcriptional regulation in these contexts is understudied. It was hypothesised that ERK relieves CIC-mediated repression downstream of FGF signalling and post-wounding during Xenopus development; this project aimed to identify targets of CIC/FGF and investigate their regulation in these contexts. Here, 44 putative CIC/FGF target genes, including the AP-1 gene fos, were identified through gene-level differential expression analysis of RNA-seq data from FGF4 overexpressing and CIC knockdown X. tropicalis embryos. Xenopus embryos were treated with FGF (SU5402), MEK (PD0325901) or nitric oxide (NO) synthase (TRIM) chemical inhibitors and the expression of fos and activated ERK investigated via in situ hybridisation and immunostaining, respectively. Gastrula stage expression of fos was found to be FGF-dependent, and CIC binding motifs were identified within a conserved region of fos intron 1. Corroborating with CIC/ERK-mediated regulation, fos expression was reduced following ERK inhibition during embryonic wound healing. Inhibiting NO production elevated activated ERK expression post-wounding, thus NO may negatively regulate fos expression via ERK. Inhibiting ERK activation similarly reduced fos expression during neurulation, suggesting comparable mechanisms may govern wound healing and morphogenesis. Other CIC targets representing AP-1 genes, namely jun and atf3, were also found to be expressed post-wounding and during neurulation. Overall therefore, the data support the hypothesis, and propose a novel role for CIC in intron mediated enhancement of the CIC/FGF target gene fos, and a wider role for CIC in regulating AP-1 gene expression during wound healing and morphogenesis.